Microbial mat compositions and localization patterns explain the virulence of black band disease in corals

Black band disease (BBD) in corals is characterized by a distinctive, band-like microbial mat, which spreads across the tissues and often kills infected colonies. The microbial mat is dominated by cyanobacteria but also commonly contains sulfide-oxidizing bacteria (SOB), sulfate-reducing bacteria (SRB), and other microbes. The migration rate in BBD varies across different environmental conditions, including temperature, light, and pH. However, whether variations in the migration rates reflect differences in the microbial consortium within the BBD mat remains unknown. Here, we show that the micro-scale surface structure, bacterial composition, and spatial distribution differed across BBD lesions with different migration rates. The migration rate was positively correlated with the relative abundance of potential SOBs belonging to Arcobacteraceae localized in the middle layer within the mat and negatively correlated with the relative abundance of other potential SOBs belonging to Rhodobacteraceae. Our study highlights the microbial composition in BBD as an important determinant of virulence.


Supplementary Tables
Supplementary Table 1

Supplementary Figure 4 Partial correlation between clr transformation matrixes of each OTU in Arcobacteraceae and BBD-progressions (a) and phylogenetic placement of 14 OTU sequences on reference tree of family Arcobacteraceae (b). (a)
The clr transformation matrixes of each OTU were calculated spearman's rank correlation with liner-progression rates and showed significant that are marked in bold with * p < 0.05 and ** p < 0.01 (a). The bubble chart showing clr transformation matrix (depicting by size) and progression rates (depicting by color). (b) The reference tree was generated from an alignment of 16S rRNA genes from a total of 653 sequences, which comprise five genera and uncultured group (b). The tree was constructed with the Maximum Likelihood method with 200 bootstrap replicates (only bootstrap values greater than 50% are denoted as black cycle at the node). For the outmost small bule star marks, it indicates that specific probe Arc94 can match the corresponding sequences (probe-matched 509 sequences). The OTUs labels with a grey star and a grey triangle on the tree indicate probe-match (a single node corresponding to a sequence that matched the probe is defined) and unclear (contains multiple nodes including miss-matched probe), respectively.
The OTUs labeled without a symbol indicate that a single node is defined but mismatched with the probe. Figure 5 Autofluorescence spectra at excitation of a laser 552 nm on an undecalcified coral section (a) and fluorescence in situ hybridization image using a negative control probe Non338 (b). Autofluorescence spectra (a) of cyanobacteria, Symbiodiniaceae and skeleton on a FISH-untreated undecalcified coral section, excited by a laser 522 nm (5% intensity), using a Lambda scan function of a confocal (TCS SP8, Leica). FISH images using the probe Non338 labeled with Cy3 showing a merged image (b) from three different channels (c-e). Given that skeleton is not shown autofluorescence, the FISH result indicating the non-specific binding on skeleton region from Non338 probe. Scale bars indicate 50 µm. To confirm the presence of cyanobacteria, SRB, SOB (except for the Rhodobacteraceae and Arcobacteraceae), and other heterotrophic bacteria that indicate BBD-microbiome reported in previous studies, we focused on the representative OTUs (operational taxonomic unit[s]) that considering compromised >1% of total relative abundance (Supplementary Table 2). The most dominant bacterial family was the cyanobacterial family Desertifilaceae, which was currently proposed as new family in order Oscillatoriales 21 , accounting for 42.57% of total relative abundance (Supplementary Table 1). In the Desertifilaceae, OTU 1 contributed predominantly or rarely to all microbial communities (in ranging from 1.38 to 77.67%) and affiliated with Roseofilum AO1-A which was isolated from BBD in Australia 2 and detected across the Indo-Pacific (Supplementary Table 2). As other dominant cyanobacteria affiliated with the family Oscillatoriaceae, OTU 23 was found in four samples from the both locations (in the ranging from 0.03 to 9.71%) with the linear-migration rates from 2.34 to 4.19 mm/day, and closely related with 98.01% similarity to Vermifilum ionodolium (accession No. MT321585.1) that retrieved from coastal limestone in USA. Interestingly, the second closest sequence (DQ446127.2) of OTU 23 according to 96.02% similarity were obtained from BBD in Bahamas (Supplementary Table 2).

Supplementary
SRB belonging to the Desulfovibrionaceae and Desulfobacteraceae, respectively represented by OTU 12 and OTU 5, showed ranged in proportions between 0.01 and 4.10% and between 0.02 and 7.98%. The OTU 12 affiliated to the Desulfovibrionaceae had a 100% similarity with an uncultured delta proteobacterium clone CD22D5 (AY497300.1) that was detected in BBD mat in the Caribbean Sea (Supplementary Table 2). The OTU 5 in Desulfobacteraceae was also closely related to uncultured bacterium Thai19_G02 (99.6% similarity), obtained from the coral disease white plague in Thailand, as the second closest lineage (Supplementary Table 2).
Besides the Rhodobacteraceae and Arcobacteraceae, we found only few reads of a common SOB member Beggiatoa spp. in our samples. In the family Beggiatoaceae, three OTUs (OTU 65, OTU 140 and OTU 815) accounted for the relative abundance ranging from 0.0018 to 0.98% in seven samples with the linear-migration rates from 2.13 to 4.85 mm/day (data not shown).
In other heterotrophic bacteria, the represented OTUs in most of families such as Vibrionaceae, Alteromonadales_unclassified, Colwelliaceae, Nitrincolaceae, Pseudoalteromonadaceae, Caminicellaceae, Lachnospiraceae, and Lachnospirales_unclassified showing varied relative abundances among our samples were linked with BBD and other coral diseases (Supplementary Table 2). Notably, OTU 7 in Vibrionaceae showed high relative abundance with 18.74% in samples with the fastest migration rates (Fig. 3 and Supplementary Table 2). Although the amplicon sequences could only provide good resolution at the genus level 3 , the best hit list of the sequences indeed included several Vibrio type strains in the blast search.